The overall aim of the application is to understand the fundamental basis of the interaction of local anesthetic/antiarrhythmic drugs (LA) with the cardiac voltage-gated sodium (Na) channel (NaV1.5). The experiments will use wild-type (WT) and site-directed mutant Na channels heterologously expressed in mammalian cells and oocytes combined with molecular modeling of the Na channel pore. Aim 1. The LA binding site in the pore. We hypothesize that binding of LA to DIV-Phe1759 directly interacts with DIIIS6 residues or indirectly by close-packed interactions between DIIIS6 and DIVS6 in the open/inactivated channel configuration that results in stabilization of the DIIIS4. In this aim we propose to further explore drug interactions with the Na channel inner pore between S6's in DIV and DIII and stabilization of their corresponding S4's. In pursuance of this hypothesis we will determine the role of DIII-Leu1461 in the coupling of DIV to DIII. Aim 2: The link between fast inactivation and LA affinity. We hypothesize that binding of the fast inactivation lid is obligatory for stabilizing the high-affinity drug/channel interaction. In this aim we propose to determine how fast inactivation contributes to high affinity LA block by correlating the size and flexibility of LA and anticonvulsant drugs to their EC50's in mutant channels with fast inactivation removed by mutagenesis of the inactivation lid and by investigating the basis of the recently reported lidocaine-induced Brugada Syndrome NaV1.5 channelopathy, V232I+L1308F. Aim 3. LA interactions with the closed channel and with early steps in activation. We hypothesize that the opening of the Na channel pore formed by the S6 segments is passive, and that drug binding to the closed channel modestly stabilizes this configuration. In pursuance of this hypothesis we will determine the residues at the bundle crossing by crosslinking Cys substitutions between S6 segments.